This is the continuation of an ongoing project in the population genetics of E. coli, the objective of which is to determine the molecular mechanisms by which amino acid substitutions in enzymes affect the activity of the enzyme and the physiology and growth rate of the organism. Amino acid substitutions are derived from three sources -- (1) naturally occurring electrophoretic variants of gnd, pgi, zwf, and other genes; (2) variants created by site-directed mutagenesis or by the reversion of nonsense codons in the phoA and lacZ genes; and (3) mutants of gnd that result from selection for faster growing strains that arise in gluconate chemostats inoculated with a strain containing an edd mutation. Functional analysis of the variant enzymes includes characterization in terms of enzyme kinetics and especially differential growth rate in chemostats in which growth is limited by the availability of key substrates. These data will permit an evaluation of the proposed saturation model of enzyme evolution, in which it is predicted that many mutations that result in small changes in enzyme activity produce negligible changes in fitness. The project includes the development of a novel type of continuous culture device which permits fluctuations in the type or concentration of nutrient, in the expectation that nonequilibrium conditions may produce different results than equilibrium conditions. The proposal also includes extensive cloning and sequencing of gnd alleles from a critical set of natural isolates in order to evaluate the clone concept of population structure and to determine the extent of nucleotide variation in structural versus regulatory regions. Molecular analysis of gnd alleles selected in chemostats will also be carried out. Data and inferences derived from the proposed experiments will be important in evaluating the effects of naturally occurring genetic variants in other organisms and in predicting the functional effects of new mutations.